Radiotherapy is used to manage pancreatic cancer; however, there is a high probability of both loco-regional relapse and metastasis. We have shown that radiation rapidly induces human pancreatic cancer cell invasion, and that it is caused by surface upregulation of activated alpha5 beta1 integrin fibronectin receptors. Our results suggest that systemic inhibition of alpha5 beta1-mediated invasion and survival might be an effective way to reduce radiation-induced pancreatic cancer cell invasion, as well as increasing the effectiveness of radiotherapy as an inhibitor of clonogenic survival and inducer of apoptosis in pancreatic cancer cells. Since we have shown that activated alpha5 beta1 receptors mediate both metastatic and angiogenic invasion, they are key to cancer progression. We devised the PHSCN peptide, Ac-PHSCN-NH2, as an inhibitor of alpha5 beta1- mediated invasion by both metastatic cancer cells and their associated microvasculature. Systemic PHSCN prevented disease progression for prolonged periods in multiple preclinical models, in our lab and in others'. PHSCN monotherapy also prevented disease progression for 4-14 months in Phase I clinical trial, without toxicity. However, because PHSCN must be administered thrice weekly as a large subcutaneous injection or infusion, a more potent form is essential for further clinical development. Recently, we found that replacing the His (H) and Cys (C) residues of the PHSCN peptide with their D-isomers, h and c, to form Ac-PhScN-NH2, yields an invasion inhibitor that is 200,000-fold more potent than PHSCN. Hence, we propose to utilize irradiated Panc-1 and BxPC-3 human pancreatic cancer cells to (1) evaluate the increase in potency of PhScN, relative to PHSCN, as an inhibitor of radiation-induced, surface alpha5 beta1 integrin and MMP1 upregulation, and basement membrane invasion in vitro; (2) evaluate the increase in potency of PhScN, relative to PHSCN, as an inhibitor of clonogenic survival and as an inducer of radiation-induced apoptosis; and (3) determine the optimal scheduling of PhScN exposure with radiation in vitro. The proposed research is necessary to allow development of PhScN as a practical systemic therapy, to be administered in combination with radiation. In support of this proposal we present the following preliminary data, in addition to our publications: 1) PhScN is 2 x 105-fold more potent than PHSCN as an inhibitor of a5b1-mediated BxPC-3 invasion; 2) PHSCN blocks radiation-induced Panc-1 invasion of basement membranes; 3) PHSCN inhibits radiation-induced surface alpha5 beta1 upregulation in Panc-1 and BxPC-3 cells; 4) PhScN exposure reduces viability, perhaps by inducing Caspase-3 activation; and 5) prior exposure to PhScN can significantly radiosensitize BxPC-3 cells. The proposed research is unique and original because it represents the first time that a fully endoproteinase- resistant agent has been utilized in pancreatic cancer. Moreover, it also represents the first time that alpha5 beta1 integrin has been targeted in pancreatic cancer. PUBLIC HEALTH RELEVANCE: Radiotherapy is used to manage pancreatic cancer; however, there is a high probability of both relapse and metastasis. Recently, we devised the PhScN peptide, which cannot be degraded by proteinases, as a highly potent inhibitor of metastatic pancreatic cancer invasion. The proposed research is necessary to allow development of PhScN as a practical systemic therapy, to be administered in combination with radiation.